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Development of lipodisks as carriers for cationic amphiphilic peptides
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. (Katarina Edwards)
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Antibiotics have made a tremendous contribution to mankind. They are one of the most successful medicines in human history. However, more and more bacterial strains develop resistance and the risk to public health can hardly be overstated. New types of antibiotics are urgently needed. Antimicrobial peptides (AMPs) have emerged as potential antibiotics because of their broad-spectrum activities and non-conventional mechanism of action. More recently, they have also received attention as promising anticancer agents. The clinical and commercial development of AMPs as a new generation of antibiotics and anticancer drugs is hampered, however, by issues concerning the toxicity, specificity and stability of the peptides.

The aim of this thesis has been to explore if formulation in a novel type of nanocarriers, referred to as lipodisks, can be used to increase the therapeutic potential of AMPs as antimicrobial and anticancer agents. Focus has been on AMPs classified as cationic amphiphilic peptides.

Encouragingly, the data presented suggests that the therapeutic potential of the AMP melittin as an antimicrobial and anticancer agent can be substantially increased by formulation in lipodisks. When formulated in the lipodisk, melittin is protected against enzymatic degradation. The lipodisk also offer a slow-release effect that sustains the bacterial cell-killing effect. We also show that specific delivery of melittin to tumour cells can be obtained by formulating the peptide in small EGF-targeting lipodisks.

Melittin contains a tryptophan residue and its interaction with lipodisks can be characterized by means of fluorimetric binding assays. In order to investigate the binding behavior also for peptides that lack intrinsic fluorescence, we developed a method based on measurements using the QCM-D technique. Studies using this, and other techniques, confirmed that it is a general behavior for cationic amphiphilic peptides to preferentially bind to the highly curved rim of lipodisks. Results of our binding studies show that the peptide to lipid ratio in the lipodisks can be tuned and optimized by varying the size and charge of the disks.

Taken together, the findings in this thesis point towards PEG-stabilized lipodisks as promising nanocarriers for antibacterial and anticancer peptides.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. , 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1442
National Category
Natural Sciences Physical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-305443ISBN: 978-91-554-9729-3OAI: oai:DiVA.org:uu-305443DiVA: diva2:1038406
Public defence
2016-12-02, B41, BMC, Husargatan 3, Uppsala, 10:15 (Swedish)
Opponent
Supervisors
Available from: 2016-11-11 Created: 2016-10-18 Last updated: 2016-11-16
List of papers
1. PEG-stabilized lipid disks as carriers for amphiphilic antimicrobial peptides
Open this publication in new window or tab >>PEG-stabilized lipid disks as carriers for amphiphilic antimicrobial peptides
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2011 (English)In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 156, no 3, 323-328 p.Article in journal (Refereed) Published
Abstract [en]

Antimicrobial peptides hold potential as a possible alternative, or complement, to conventional antibiotics but new, safe and efficient means are needed for formulation and administration of the peptides. In this study we have investigated the utility of a novel type of lipid particles, the polyethylene glycol-stabilized lipid-disks, as carriers for the model peptide melittin. The structural integrity of the carrier particle when loaded with the peptide was investigated using cryo-transmission electron microscopy. Liposome leakage upon addition of the peptide-lipid-disks was monitored as a means to verify the membrane lytic effect of the formulation. The susceptibility of melittin to tryptic digestion was studied and compared in the absence and presence of lipid-disks. Finally, the antibacterial effect of the peptide-lipid-disk formulation was compared to that of free melittin after both single and repeated exposure to Escherichia coli. The results show that melittin can redistribute from the disk into a new host membrane and that formulation in the disks does not compromise melittin's membrane permeabilizing ability. Further, the peptide was found to be fully protected against degradation when bound to the disks. Time-kill experiments revealed that all the antibacterial effect of melittin administered in free form was gone after a single exposure to E. coli. In contrast, the disk formulation showed significant cell-killing effect also upon a second exposure to bacteria, indicating an extended release of peptide from the lipid-disks. These results suggest that the lipid-disks constitute a new class of promising carriers for peptide antibiotics.

Keyword
PEG-stabilized lipid disk, Antimicrobial peptide, Melittin, E. coli, Peptide delivery
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-158960 (URN)10.1016/j.jconrel.2011.08.029 (DOI)000298555000007 ()21903146 (PubMedID)
Available from: 2011-09-19 Created: 2011-09-19 Last updated: 2016-10-18Bibliographically approved
2. Effect of α-helical peptides on liposome structure: A comparative study of melittin and alamethicin
Open this publication in new window or tab >>Effect of α-helical peptides on liposome structure: A comparative study of melittin and alamethicin
2010 (English)In: Journal of Colloid and Interface Science, ISSN 0021-9797, E-ISSN 1095-7103, Vol. 346, no 1, 127-135 p.Article in journal (Refereed) Published
Abstract [en]

Cryo-transmission electron microscopy was used in combination with turbidity and leakage measurements to explore and compare the membrane perturbing effects of melittin and alamethicin on POPC-based liposomes of varying composition. The results show that the two peptides, despite their differences in physico-chemical properties and proposed mode of action, induce similar structural effects on the liposomes. Importantly, whereas low peptide concentrations leave pure POPC liposomes intact and seemingly unperturbed, POPC liposomes supplemented with 40 mol.% cholesterol change their shape, rupture and fuse in response to the addition of both melittin and alamethicin. In the case of alamethicin, but not melittin, fusion is effectively prevented by inclusion of 10 mol.% POPG in the liposome membranes. By means of a competitive binding assay we furthermore show that alamethicin, in line with earlier findings for melittin, possess high affinity for positively curved lipid surfaces. Moreover, results from the present study show that magainin 2 has a similar preference for curved surfaces.

Place, publisher, year, edition, pages
Elsevier, 2010
Keyword
Melittin, Alamethicin, Magainin, Liposomes, Affinity, Cryo-transmission electron microscopy
National Category
Physical Chemistry
Research subject
Chemistry with specialization in Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-124618 (URN)10.1016/j.jcis.2010.02.032 (DOI)000277132700020 ()20226468 (PubMedID)
Available from: 2010-05-05 Created: 2010-05-05 Last updated: 2016-10-18Bibliographically approved
3. EGF-targeting lipodisks for specific delivery of cationic amphiphilic peptides to tumour cells
Open this publication in new window or tab >>EGF-targeting lipodisks for specific delivery of cationic amphiphilic peptides to tumour cells
(English)Manuscript (preprint) (Other academic)
National Category
Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-305375 (URN)
Available from: 2016-10-17 Created: 2016-10-17 Last updated: 2016-11-11
4. Characterizing and controlling the loading and release of cationic amphiphilic peptides onto and from PEG-stabilized lipodisks
Open this publication in new window or tab >>Characterizing and controlling the loading and release of cationic amphiphilic peptides onto and from PEG-stabilized lipodisks
2016 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827Article in journal (Refereed) Published
Abstract [en]

Recent studies have identified PEG-stabilized lipid nanodisks (lipodisks) as promising carriers for cationic amphiphilic peptides with antimicrobial and anticancer activity. Using fluorimetric and nanogravimetric methods, we have in this work characterized the parameters describing and controlling the binding of three selected peptides (melittin, LL37, and magainin 2) onto lipodisks. It was found that the affinity of melittin for lipodisks is independent of the disk size and rim charge. On the other hand, the number of binding sites is strongly dependent on both parameters, with the highest loading being obtained for small disks with a negatively charged rim. An optimized composition of the lipodisks was utilized to study the loading of antimicrobial peptides magainin 2 and human LL37. It was observed that although magainin 2 can be loaded in large amounts, it is released very fast upon dilution, which limits future therapeutic applications. In contrast, LL37 can be loaded at relevant concentrations and the formulation is stable. This opens up for applications of LL37-loaded lipodisks as antibiotics and in anticancer treatments.

National Category
Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-305377 (URN)10.1021/acs.langmuir.6b03012 (DOI)
Funder
Swedish Cancer Society
Available from: 2016-10-17 Created: 2016-10-17 Last updated: 2016-11-11Bibliographically approved
5. Label-Free Characterization of Peptide-Lipid Interactions Using Immobilized Lipodisks
Open this publication in new window or tab >>Label-Free Characterization of Peptide-Lipid Interactions Using Immobilized Lipodisks
2013 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 85, no 15, 7377-7384 p.Article in journal (Refereed) Published
Abstract [en]

Lipodisks, planar lipid bilayer structures stabilized by PEG-ylated lipids, were in the present study covalently bound and immobilized onto sensors for quartz crystal microbalance with dissipation monitoring (QCM-D) studies. It is shown that the modified sensors can be used to characterize the interaction of lipodisks with α-helical amphiphilic peptides with an accuracy similar to that obtained with well established fluorimetric approximations. The method presented has the great advantage that it can be used with peptides in their native form even if no fluorescent residues are present. The potential of the method is illustrated by determining the parameters describing the association of melittin, mastoparan X, and mastoparan with immobilized lipodisks. Both thermodynamic and kinetic analyses are possible. The presented method constitutes a useful tool for fundamental studies of peptide–membrane interactions and can also be applied to optimize the design of lipodisks, for example, for sustained release of antimicrobial peptides in therapeutic applications.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-205638 (URN)10.1021/ac4012842 (DOI)000323014000056 ()
Available from: 2013-08-21 Created: 2013-08-21 Last updated: 2016-10-18Bibliographically approved

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